The invention relates to a method for separating detection signals in the beam path of an optical device, different signals being formed in a defined temporal sequence.
In quite general terms, what is involved here is a method for separating detection signals in the beam path of an optical device irrespective of the particular application/use. It is essential here that detection signals of different origin and quality are formed in a temporal sequence. The optical device can be, for example, a fluorescence microscope, in particular a confocal laser microscope. Biological samples are marked with appropriate dyes for use in fluorescence microscopy. Said dyes are usually excited to emission with laser light. The fluorescent light is detected for the purpose of imaging.
Reflections which are formed directly on the sample surface and in the transition of the excitation light from one optical medium to another, for example from air to glass or vice versa, are disadvantageous. In the final analysis, interfering scattered light is produced here.
Furthermore, there is the problem that the fluorescent light is approximately 1 million times weaker than the exciting laser light.
This means that for imaging purposes it is essential to separate the reflected excitation light and/or scattered light and fluorescent light and/or their signals as efficiently as possible from one another.
It may be noted at this juncture that the fundamental concern here is the separation of detection signals in the beam path of an optical device, and that fluorescence microscopy is mentioned merely by way of example. The inventive method can therefore always be applied when what is involved is the separation of “mixed” detection signals which are formed in a defined temporal sequence.
Different methods are known from practice for separating the excitation/scattered light from the fluorescent light required for imaging or evaluation, that is to say for separating their signals, in the case of the previously mentioned example, that is to say in fluorescence microscopy. Beam splitters or acousto-optical elements are used for this purpose in the detection beam path. Use is made of the possibility of distinguishing excitation/scattered light and fluorescent light by means of their mutually shifted wavelengths. It is essential in this case that the fluorescent light usually has a larger wavelength than the scattered light.
Again, it is already customary in practice to minimize the undesired reflections by means of an antireflection coating on optical components, in particular on glasses. This—passive—measure permits the reduction of the proportion of the reflected light from a few percent to a few tenths of a percent. However, this is not sufficient.
Furthermore, it is known from practice to suppress the undesired reflected light by means of optical notch filters. These filters likewise use the shifting of the wavelengths in the case of reflected/scattered light and fluorescent light. When notch filters are used, suppression of the reflected/scattered light reaches an optical density of to 8 and more, an optical density of 7 corresponding to a transmission of approximately 0.00001%. At the same time, the transmission of the fluorescent light is up to or over 99%.